Complete Title of Thesis:
"A new model to explain asymmetric poliovirus RNA replication revealed by studies of preinitiation replication complexes and CRE(2C)-dependent VPg uridylylation."
Prepared under the direction of: Dr. David J. Barton Ph.D.
Poliovirus RNA replication occurs via a multistep process. Upon its release into the cytoplasm of a susceptible cell, genomic RNA is first translated into viral proteins and then replicated within membranous replication complexes. Within membranous RNA replication complexes the viral RNA is copied into a complementary negative-strand RNA that then serves as a template for the asymmetric synthesis of positive-strand RNAs, identical to the genomic RNA. Nascent positive-strand RNAs can either amplify the replicative cycle by serving as mRNAs or be packaged into virions.
The mechanism(s) regulating poliovirus RNA replication remain obscure. Results from our lab and others have shown that RNA structures from both the 5' and the 3' nontranslated regions are coordinately required for the synthesis of negative-strand RNA. These results have inspired numerous models in which the specific replication of poliovirus RNA is regulated by genome circularization, mediated by 5' -3' terminal RNP interactions. To further investigate the nature of poliovirus RNA replication we used cell-free translation-replication reactions and preinitiation replication complexes (PIRCs). Poliovirus RNA translates and replicates authentically and efficiently within cell-free translation replication reactions.
In this investigation mutant RNA templates were characterized for RNA replication within PIRCs. Contrary to previous reports, the IRES was completely dispensable for both negativeand positive-strand RNA synthesis. While the IRES was not directly involved in RNA replication, interaction of the viral RNA with the translation machinery potentiated efficient RNA replication. These data indicate that the 5' and 3' terminal RNP interactions required for viral RNA replication may be imposed initially by the cellular translation machinery. Our investigations also demonstrated that in addition to negative-strand RNA synthesis, the 5' terminal cloverleaf was also required for the CRE(2C)-dependent uridylylation of viral protein 3B (VPg). Our experimental data indicate that CRE(2C)-dependent VPg uridylylation was dispensable for negative-strand RNA synthesis but was absolutely required for positive-strand RNA synthesis. These data provide the basis for a new model of poliovirus RNA replication. This new model emphasizes long range interactions between the 5' cloverleaf, 3' NTR and CRE(2C), and provides mechanistic insight into the specific and asymmetric nature of poliovirus RNA replication.